U.S. patent application number 11/443734 was filed with the patent office on 2006-12-07 for method for optically determining dynamic behaviour of contracting muscle cells.
This patent application is currently assigned to Fraunhofer-Gesellschaft zur Foerderung der angewandten Forschung e.V.. Invention is credited to Ron Schwarz.
Application Number | 20060275745 11/443734 |
Document ID | / |
Family ID | 35169982 |
Filed Date | 2006-12-07 |
United States Patent
Application |
20060275745 |
Kind Code |
A1 |
Schwarz; Ron |
December 7, 2006 |
Method for optically determining dynamic behaviour of contracting
muscle cells
Abstract
The invention refers to a method for optically determining the
dynamic behaviour of contracting muscle cells, such as
cardiomyocytes in a cell culture, for example, wherein a video
sequence of the contracting muscle cells is made using a video
camera (3), an image processing unit (4) is used to calculate a
displacement vector for each image element and each moment of the
video sequence, said vector describing the movement of the image
content in the image element, and at least one parameter describing
the dynamic behaviour of the muscle cells is determined from the
displacement vectors.
Inventors: |
Schwarz; Ron; (Duesseldorf,
DE) |
Correspondence
Address: |
SHUMAKER & SIEFFERT, P. A.
8425 SEASONS PARKWAY
SUITE 105
ST. PAUL
MN
55125
US
|
Assignee: |
Fraunhofer-Gesellschaft zur
Foerderung der angewandten Forschung e.V.
Muenchen
DE
|
Family ID: |
35169982 |
Appl. No.: |
11/443734 |
Filed: |
May 31, 2006 |
Current U.S.
Class: |
435/4 ; 382/128;
600/315; 702/19 |
Current CPC
Class: |
G06K 9/0014 20130101;
G06T 7/0012 20130101; G06T 2207/30024 20130101; G06T 7/20
20130101 |
Class at
Publication: |
435/004 ;
382/128; 702/019; 600/315 |
International
Class: |
C12Q 1/00 20060101
C12Q001/00; G06F 19/00 20060101 G06F019/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 1, 2005 |
EP |
05 104 713.2 |
Claims
1. A method for optically determining the dynamic behaviour of
contracting muscle cells, the method comprising: obtaining a video
sequence of the contracting muscle cells is made using a video
camera (3), and using an image processing unit (4) to calculate a
displacement vector for each image element and each time step of
the video sequence, said vector describing the movement of the
image content in the image elementa, wherein at least one parameter
describes the dynamic behaviour of the muscle cells is determined
from the displacement vectors.
2. The method of claim 1, wherein at least one parameter describes
the contraction rate, the amplitude of the displacement or the
contractility.
3. The method of claim 1 or 2, wherein the displacement vectors are
determined using non-rigid image registration methods.
4. The method of one of claims 1 to 3, wherein moments of the
reversal of the direction of movement are determined from the
displacement vectors.
5. The method of one of claims 1 to 4, wherein a parameter
describing contractility is calculated from the vector field
divergence of the displacement vectors.
6. The method of claims 1 to 5, wherein the contracting muscle
cells include cardiomyocytes in a cell culture.
7. A system for optically determining the dynamic behaviour of
contracting muscle cells, the system comprising: means for
obtaining a video sequence of the contracting muscle cells using a
video camera (3), and an image processing unit (4) that calculates
a displacement vector for each image element and each time step of
the video sequence, said vector describing the movement of the
image content in the image element, wherein at least one parameter
describing the dynamic behaviour of the muscle cells is determined
from the displacement vectors.
8. The system of claim 7, wherein at least one parameter describes
the contraction rate, the amplitude of the displacement or the
contractility.
9. The system of claim 7, wherein the displacement vectors are
determined using non-rigid image registration methods.
10. The system of claim 7, wherein moments of the reversal of the
direction of movement are determined from the displacement
vectors.
11. The system of claim 7, wherein a parameter describing
contractility is calculated from the vector field divergence of the
displacement vectors.
12. The system of claim 7, wherein the contracting muscle cells
include cardiomyocytes in a cell culture.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention is directed to a method for optically
determining the dynamic behaviour of a cell culture including
contracting muscle cells and preferably cardiomyocytes.
[0003] 2. Description of the Prior Art
[0004] Recent years have seen serious progress in stem cell
research. Thus, stem cell based samples can be used to replace
animal tests in the development of new drugs. As a substitute for
experiments on explanted animal hearts, in-vitro experiments on
cardiomyocytes differentiated from stem cells can be performed. The
dynamic behaviour of cell cultures, i.e. the contraction behaviour
of the cardiomyocytes, gives information about the toxic effect of
substances added. The contraction behaviour can be studied and
quantified by optical monitoring via a microscope.
[0005] There exist several approaches to determine the dynamic
behaviour of con-tracting cells. The classical approach is based on
human inspection of cells in a culture. For large scale
experiments, this does no longer work and automatic approaches are
required. Advanced systems use cameras to capture time sequences of
images from cells in a culture. Subsequently, image analysis
methods are applied to the image sequence to derive cell parameters
that describe the dynamic behaviour.
[0006] In prior art, substances are examined for their
cardiotoxicity by making experiments on Langendorff preparations.
Here, an animal heart is explanted and perfused. Parameters such as
the ventricular contraction force, the heart rate and the coronary
blood flow are measured at the beating heart, and the variations of
the parameters occurring when substances are administered are
measured. These experiments have the great disadvantage that they
require the killing of animals to obtain the hearts needed to
perform the experiment. They are costly and require an elaborate
preparation of animal organs.
[0007] Recent developments use methods based on cell cultures to
perform cardio toxicity tests. Here, cell culture dishes are
employed that are provided with a plurality of electrodes.
Cardiomyocytes are cultivated in these dishes, which contract
spontaneously without any external excitation. The electrodes allow
to derive electric signals with which electro-physiologic
parameters of the contracting cardiomyocytes can be determined
(see, for example, WO-A-2004/067734). However, the contraction
force of the cardiomyocytes can not be determined by this
method.
[0008] Current approaches for estimation of contraction parameters
look at local features detected in an image, try to identify the
same feature in subsequent images, and derive movement parameters
of the detected image element. In EP 1 302 535 A1 it is proposed to
look at areas with low or high intensity and monitor their
movements. According to the article of Weisensee D et al: In vitro
Approach to `Uremic Cardiomyopathy`. Nephron 65 (1993) 392-400 and
Riehle M and Bereiter-Hahn J: Ouabain and Digitoxin as Modulators
of Chick Embryo Cardiomyocyte Energy Metabolism.
Arzneim.-Forsch./Drug Res. 44(II), 8 (1994) 943-947 difference
images showing all pixels which have changed as an indication of
movements are generated. According to Gervais-Pingot V et al: In
vitro quantification by image analysis of inotropic and
chronotropic effects of drugs on cultures of cardiac myocytes. Cell
Biology and Toxicology 10 (1994) 297-300 optical densities of an
analysed area are monitored and contraction parameters from changes
in density are derived. According to Korohoda W et al: A new model
for the research into rhythmic contraction activity of
cardiomyocytes in vitro. Biochemistry and Cell Biology 73, 7-8
(1995) 431-439 changes of the size of a black area at a fixed
position within the image sequence are considered.
[0009] The approach of the present invention uses non-rigid
registration methods to compare subsequent images. These
registration methods are generally used for geometric alignment of
images (e.g. of electrophoresis gels with different gene
expressions) or volume data from different sources (e.g. CT, MR) or
at different times (e.g. pre-operative/post-operative) for
comparison reasons. When computing a registration transformation,
the whole image is inspected and a mapping between two images is
generated which maps the whole image optimally rather than only a
specific image part. When more than one image element moves, the
registration approach has the advantage that all moving parts are
taken in account and improve the precision for a single element
whereas in local approaches other moving image elements can and
will disturb the calculation of the selected element.
[0010] Some approaches for non-rigid registration use displacement
fields to model image deformations and find the optimal
registration transformation, e.g. Lau Y H et al.: Non-rigid image
registration using median-filtered coarse-to-fine displacement
field and a symmetric correlation ratio. Phys. Med. Biol. 46 (2001)
1297-1319. The displacement field, that is an intermediate means in
establishing a non-rigid transformation, is the crucial result for
the approach of the present invention where displacement fields are
used to estimate movements in image sequences in a more stable and
more precise manner than local methods can achieve.
[0011] EP-A-0414237 describes a device for analyzing the change of
the shape of individual cardiomyocytes caused by contraction and a
change in the intercellular concentration of calcium ions. Here,
the signal of a fluorescent dye is detected optically.
[0012] The mechanical measurement of the contractility of
individual cardiomyocytes is described in SU-A-1377738. Here, a
mechanical fixation of individual cells is required.
SUMMARY OF THE INVENTION
[0013] The invention allows for an optical determination of the
contraction behaviour of cardiomyocytes in a cell culture. Thus,
the effects of test substances on cardiomyocytes can be studied in
vitro.
[0014] The invention provides a method for optically determining
the dynamic behaviour of a cell culture of contracting muscle
cells, such as cardiomyocytes in a cell culture, wherein [0015] a
video sequence of the contracting muscle cells is made using a
video camera, [0016] an image processing unit is used to calculate
a displacement vector for each image element and each time step of
the video sequence, said vector describing the movement of the
image content in the image element, and [0017] at least one
parameter describing the dynamic behaviour of the muscle cells is
determined from the displacement vectors.
[0018] Thus, according to the invention, the movement of the
contracting muscle cells is recorded with a video camera. The video
camera produces a video sequence. Using image processing methods,
the changes in the contracting muscle cells in each image element
are determined, from which displacement vectors are calculated that
describe the movement of the image content. The displacement
vectors allow to obtain at least one parameter describing the
dynamic behaviour of the muscle cells. This parameter may be, for
example, the contraction rate, the amplitude of the displacement
and/or the contractility, i.e. the force with which the muscle
cells contract.
[0019] In order to determine the dynamic behaviour of the
contracting muscle cells from the video sequence the video images
need to be registered to a reference image which is selected from
the sequence. Alternatively each two consecutive images can be
registered. All images are matched using a non-rigid hierarchical
registration method. The registration is based on a uniform grid
which is used for sub-division of the images. The images are warped
by shifting individual grid points and by interpolating the images
between them. The warped images are compared to the reference image
using a similarity measure (e.g. correlation) which determines the
matching quality of the warped and the reference image. Standard
optimization techniques are used to maximize the similarity
measure. The successive refinement of the grid results in a
non-rigid registration of each image to the references image,
yielding vector fields of local displacements which reflect the
movement of the cells at each time step.
[0020] The vector fields are used for derivation of several
parameters describing the movement of the muscle cells. By
detecting points of return of the movement direction parameters
such as frequency and phase shift of contraction can be derived.
The vector field divergence can be calculated to provide a measure
that detects changes in the density of the displacement vector
field, i.e. expansion and relaxation of muscle cells.
[0021] Thus, the main feature of the invention is the recording of
movement images and the analysis thereof for a quantitative
determination of at least one parameter describing the dynamic
behaviour of the muscle cells.
[0022] Improvements and advantages over prior art provided by the
invention
[0023] The method of the present invention has the following
advantages over prior art. It allows to determine the contractility
of cardiomyocytes in a cell culture. The measuring is not
restricted to individual cardiomyocytes, but allows for a measuring
in a group of cells. No special cell culture dishes with electrodes
are required. The method determines the actual movement behaviour
of the cardiomyocytes. It needs no addition of dyes and is
non-invasive, i.e. the cells are not influenced by the method. No
animals have to be killed. No complicated preparation of the
analyzed samples is required. The method allows for an automated
analysis of cell cultures and is thus suited for performing test
series with a high throughput.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The following is a description of the invention with
reference to the drawing for the case that the contracting cells
are cardiomyocytes, wherein the drawing shows a test structure for
optically determining the dynamic behaviour of contracting muscle
cells.
DESCRIPTION OF PREFERRED EMBODIMENT
[0025] In the embodiment, the optical determination of the dynamic
behaviour of the cell culture of contracting cardiomyocytes is
performed according to the following steps: [0026] cell cultures
with contracting cardiomyocytes are recorded under a microscope 2
using a video camera 3, [0027] video sequences of the contracting
cardiomyocytes recorded by the video camera 3 are supplied to a
computer (image processor) 4, [0028] a software analyzes the
movement of the cardiomyocytes in the video sequences and
determines a displacement vector for each image element and each
time step of the video sequence, the displacement vector describing
the movement of the image content in the image element, [0029] at
least one parameter describing the dynamic behaviour of the
cardiomyocytes is quantitatively determined from the displacement
vectors, this parameter being, in particular, the contraction rate,
the amplitude of the displacement and/or the contractility.
[0030] Cardiomyocytes are differentiated from stem cells and are
cultured in a cell culture dish 1. A microscope 2 is connected to a
video camera 3 which supplies the video image to a computer 4. The
contracting cardiomyocytes are recorded by the video camera 3 for a
determined period of time and the video data are stored on the
computer 4.
[0031] A reference image is selected from the video data and each
single recorded image is geometrically aligned with the reference
image using a non-rigid registration method. Thus, a local
displacement vector field of the individual image elements is
calculated for each video image. This field describes the movement
state of the cardiomyocytes at any moment.
[0032] The moments of the beginning of the contraction phase and of
the maximum excursion are determined for each image element in the
sequence of displacement vectors. Thereafter, the parameters of the
contraction rate and the amplitude of displacement can be
calculated.
[0033] Contractility, i.e. the force with which the cardiomyocytes
contract, is determined as another parameter of the dynamic
behaviour of the cardiomyocytes. For each image element, the vector
field divergence of the displacement vector field between the
moment of the beginning of a contraction phase and the moment of
maximum excursion is calculated. By adding these divergences, the
contractility is obtained.
[0034] Although the invention has been described and illustrated
with reference to specific illustrative embodiments thereof, it is
not intended that the invention be limited to those illustrative
embodiments. Those skilled in the art will recognize that
variations and modifications can be made without departing from the
true scope of the invention as defined by the claims that follow.
It is therefore intended to include within the invention all such
variations and modifications as fall within the scope of the
appended claims and equivalents thereof.
* * * * *